Method for predicting the response to her2-directed therapy

ABSTRACT

This invention provides methods for determining or predicting response to HER2-directed therapy in an individual.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 13/945,568,filed Jul. 18, 2013, now pending; which is a continuation of U.S. patentapplication Ser. No. 13/025,600, filed Feb. 11, 2011, issued as U.S.Pat. No. 8,512,967 on Aug. 20, 2013; which is a divisional of U.S.patent application Ser. No. 10/735,118, filed Dec. 11, 2003, nowabandoned; which is a continuation-in-part of U.S. patent applicationSer. No. 10/408,520, filed Apr. 7, 2003, now abandoned, which claims thebenefit of U.S. Provisional Application No. 60/370,473, filed Apr. 5,2002. U.S. patent application Ser. No. 10/735,118 also claims thebenefit of U.S. Provisional Application No. 60/432,942, filed Dec. 11,2002. Each of these applications are herein incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for predicting the response toHER2-directed therapy in an individual.

2. Background of the Invention

Cellular growth and differentiation processes involve growth factorsthat exert their actions through specific receptors expressed in thesurfaces of responsive cells. Ligands binding to surface receptors, suchas those that carry an intrinsic tyrosine kinase activity, trigger acascade of events that eventually lead to cellular proliferation anddifferentiation (Carpenter et al., 1979, Biochem., 48: 193-216; Sachs etal., 1987, Cancer Res., 47: 1981-1986). Receptor tyrosine kinases can beclassified into several groups on the basis of sequence similarity anddistinct features. One of these groups includes the epidermal growthfactor receptor family, which includes erbB-1 (also termed EGFR orHER-1) (Carpenter et al., supra); erbB-2 (HER-2/neu) (Semba et al.,1985, Proc. Natl. Acad. Sci., 82: 6497-6501; Coussens et al., 1985,Science, 230: 1130-1139; Bargmann et al., 1986, Cell, Vol. 45, 649-657);erbB-3 (HER-3) (Kraus et al., 1989, Proc. Natl. Acad. Sci., 86:9193-9197; Carraway et al., 1994, J. Biol. Chem., 269: 14303-14306), anderbB-4 (HER-4) (Plowman et al., 1993, Nature, 366: 473-475; Tzahar etal., 1994, J. Biol. Chem., 269: 25226-25233).

Most tumors of epithelial origin express multiple erbB (HER) receptorsand co-express one or more EGF-related ligands suggesting thatautocrine-receptor activation plays a role in tumor cellularproliferation. Because these ligands activate different erbB/HERreceptors, it is possible that multiple erbB receptor combinations mightbe active in a tumor, a characteristic that could influence its responseto an erbB-targeted therapeutic. erbB receptors form homodimers andheterodimers that can be stimulated by various ligands leading todownstream signaling events, the extent and nature of which depend onthe combination of specific dimers and ligands. For example, HER2/neuappears to be the preferred heterodimerization partner with othermembers of the epidermal growth factor receptor family, but ultimatelythe dimers formed are determined by the ligand and the erbB receptorsexpressed in the cell. Not only may the ligand select the dimerizationpartners, but it may also influence the time course of membranetranslocation, activation, and internalization of the receptor. Forexample, NDF/Heregulin can stimulate tyrosine phosphorylation of erbB-2through heterodimerization with either receptors erbB-3 or erbB-4 (Peleset al., 1992, Cell 69, 205-216, Peles et al., 1993, EMBO J. 3, 961-71,Holmes et al., 1992, Science 256, 1205-1210; Tzahar et al., 1994, Biol.Chem., 269, 25226-25233; Plowman et al., 1993, Nature 366, 473-475;Pinkas-Kramarski et al., 1994, Proc. Natl. Acad. Sci. USA, 91,9387-9391; Pinkas-Kramarski et al., 1996, J. Biol. Chem., 271,19029-19032; Pinkas-Kramarski et al., 1998, Oncogene, 16, 1249-1258).Depending on the cell line studied, NDF/Heregulin can either elicit agrowth arrest and differentiation phenotype, resulting in morphologicalchanges, induction of lipids, and expression of intracellular adhesionmolecule-1; or it can induce a mitogenic response (Holmes et al., 1992,Science, 256:1205-1210; Peles et al., 1992, Cell, 69:205-216; Bacus etal., 1993, Cancer Res. 53:5251-5261).

Downstream signaling after ligand binding may be determined by the setof docking proteins that may bind to the activated receptors. Forexample, activation of erbB receptor heterodimers is coupled to andstimulates downstream MAPK-Erk1/2 and PI3K-AKT growth and survivalpathways, whose deregulation in cancer has been linked to diseaseprogression and refractoriness to therapy (Tzahar et al., 1996, Mol.Cell. Biol. 16, 5276-5287; Fukazawa et al., 1996, J. Biol. Chem. 271,14554-14559, Olayioye et al., 1998, Mol. Cell. Biol. 18, 5042-5051;Lange et al., 1998, J. Biol. Chem. 273, 31308-31316; Hackel et al.,1999, Curr. Opin. Cell Biol. 11, 184-189). HER-3 is a major docking sitefor phosphoinositide-3-kinase (PI3K). In addition, NDF/Heregulinstimulation causes activation of the PI3K pathway and phosphorylation ofAKT (Altiok et al., 1999, J. Bio. Chem. 274, 32274-32278; Liu et al.,1999, Biochem. Biophys. Res. Comm. 261 897-903; Xing et al., 2000,Nature, Med. 6 189-195). These observations implicate PI3K/AKT in thesignaling cascade that results from HER-3 heterodimerization withoverexpressed HER-2/neu receptors in breast cancer cells; activation ofPI3K/AKT promotes cell survival and enhanced tumor aggressiveness (Bacuset al., 2002, Oncogene 21, 3532-3540). In addition, AKT2 was reported tobe activated and overexpressed in HER-2/neu-overexpressing breastcancers (Id.).

erbB-2/HER-2 is overexpressed in 20 to 30% of all breast cancers, andits overexpression is associated with poor prognosis, suggesting that itcould be used as a target for anti-tumor agents (Slamon et al., 1987;Hudziak et al., 1989; Tagliabue et al., 1991). In erbB-2-overexpressingbreast cancer cells, treatment with antibodies specific to HER-2/erbB-2in combination with chemotherapeutic agents (such as cisplatin,doxorubicin, and taxol) elicits a higher cytotoxic response thantreatment with chemotherapy alone (Hancock et al., 1991; Arteaga et al.,1994; Pietras et al., 1994). One possible mechanism by whichHER-2/erbB-2 antibodies might enhance cytotoxicity to chemotherapeuticagents is through the modulation of the HER-2/erbB-2 protein expression(Bacus et al., 1992 & 1993; Stancovski et al., 1991; Klapper et al.,1997 & 2000), or by interfering with DNA repair (Arteaga et al., 1994 &2001; Pietras et al., 1994).

Because of the effect of anti-HER-2/erbB-2 antibodies on cellulargrowth, a number of approaches have been used to therapeutically targetHER-2/erbB-2- or EGFR-overexpressing cancers. For clinical use, oneapproach is to interfere with the kinase activity of the receptor byusing inhibitors that block the nucleotide binding site of HER-2/neu orEGFR (Bruns et al., 2000; Christensen et al, 2001, Erlichman et al.,2001, Herbst et al., 2002; Hidalgo et al, 2001; Moasser et al, 2001;Fujimura et al., 2002; Normanno et al., 2002). A second approach isusing ansamycins to influence the stability of HER2/neu receptors(Munster et al., 2002; Basso et al, 2002). Another approach is the useof antibodies directed to various erbB receptors, specifically EGFR orHER-2/neu (Alaoui-Jamali et al., 1997; Albanell et al., 2001(a); Baselgaet al., 1994 & 2002; Mendelsohn, 1990). Analysis of various antibodiesto HER-2/neu led to the identification of the murine monoclonal, 4D5.This antibody recognizes an extracellular epitope (amino acids 529 to627) in the cysteine-rich II domain that resides very close to thetransmembrane region. Treatment of breast cancer cells with 4D5partially blocks NDF/heregulin activation of HER-2-HER-3 complexes, asmeasured by receptor phosphorylation assays. To allow for chronic humanadministration, murine 4D5 was fully humanized to generatetrastuzumab/HERCEPTIN® (Sliwkowski et al., 1999; Ye et al.., 1999;Carter et al, 1992; Fujimoto-Ouchi et al, 2002; Vogel, et al., 2001 &2002).

A number of monoclonal antibodies and small molecule, tyrosine kinaseinhibitors targeting EGFR or erbB-2 have been developed. For example,HERCEPTIN® is approved for treating the 25% of women whose breastcancers overexpress erbB-2 protein or demonstrate erbB-2 geneamplification (Cobleigh et al., 1999, J. Clin. Oncol. 17, 2639-2648). Inaddition, several EGFR-targeted therapies are currently under clinicalinvestigation (Mendelsohn & Baselga, 2000, Oncogene 19, 6550-6565; Xiaet al., 2002, Oncogene 21, 6255-6263).

The development of successful oncological drugs has followed awell-established evaluation process including phase I, II, and IIIclinical trials. Phase I studies aim to determine the maximallytolerated dose of the drug, its optimal schedule of administration andthe dose-limiting toxicities. Historically, cytotoxic cancer therapieshave been developed based on maximum tolerated doses (MTD), treatingpatients without understanding the tumor profile for likely responders.Hence, patients were often subjected to toxic therapies with limitedtherapeutic benefit. Recently, elucidating tumor growth and survivalpathways has led to the development of tumor-targeted therapies. Forsuch targeted therapeutics that are not expected to produce seriousadverse side effects, relying on a MTD may not be suitable. Morerelevant may be the determination of the optimal dose and schedule thatis sufficient to inhibit cellular signaling in patient samples.Biological assays for signaling biomarkers are needed to establish sucha protocol.

Preclinically, most erbB-receptor targeted therapies primarily exertcytostatic anti-tumor effects, necessitating their chronicadministration in clinical practice. Identification of biologicallyeffective doses (BED), the dose or dose range that maximally inhibitsthe intended target, beyond which dose escalation is likely to addtoxicity without benefit, is therefore essential. Moreover, many ofthese agents will be used in combination with cytotoxic therapies, whereadded toxicity may not be tolerable, further supporting BED-baseddosing. “Targeted—therapy” implies that populations of likely respondersexist, and can be identified.

In view of the severe and deleterious consequences of administering aninappropriate or ineffective therapy to a human cancer patient, thereexists a need in the art for predicting the response to cancer therapyin an individual. Further, there is a need to develop diagnostics thatare capable of predicting patient response for the successfuldevelopment and clinical acceptance of new HER-2 targeted therapeuticssimilar to HERCEPTIN®.

SUMMARY OF THE INVENTION

This invention provides methods for predicting a response of anindividual to a HER2-directed therapy.

In a first aspect, the invention provides methods for identifying amammalian tumor that responds to a HER2-directed therapy, wherein themammalian tumor overexpresses HER2, the method comprising the step ofassaying a sample obtained from the mammalian tumor to detect a patternof expression, phosphorylation or both expression and phosphorylation ofone or a plurality of polypeptides consisting of:

(a) IGFR polypeptide;

(b) EGFR polypeptide;

(c) NDF polypeptide;

(d) phosphorylated S6 ribosomal polypeptide;

(e) phosphorylated AKT polypeptide; and

(f) phosphorylated ERK polypeptide;

wherein the particular combination of polypeptides and pattern ofexpression, phosphorylation or both expression and phosphorylationidentifies mammalian tumors that respond to a HER2-directed therapy.

In certain embodiments, the pattern that identifies a mammalian tumor asresponding is decreased expression of IGFR polypeptide in the mammaliantumor as compared to a non-tumor tissue or cell sample. In otherembodiments, the detected pattern is normal or increased expression ofIGFR polypeptide, accompanied by decreased phosphorylation of AKTpolypeptide, decreased phosphorylation of S6 ribosomal polypeptide orboth in the mammalian tumor as compared to a non-tumor tissue or cellsample. In further embodiments, the detected pattern is normal orincreased expression of EGFR polypeptide, accompanied by decreasedphosphorylation of ERK polypeptide in the mammalian tumor as compared toa non-tumor tissue or cell sample. In additional embodiments, thedetected pattern is decreased expression of IGFR polypeptide,accompanied by increased phosphorylation of S6 ribosomal polypeptide inthe mammalian tumor as compared to a non-tumor tissue or cell sample. Inother embodiments, the detected pattern is decreased expression of IGFRpolypeptide, accompanied by increased expression of NDF polypeptide inthe mammalian tumor as compared to a non-tumor tissue or cell sample;where further the detected pattern can include increased phosphorylationof S6 ribosomal polypeptide.

In a second aspect, the invention provides methods for identifying amammalian tumor that does not respond to a HER2-directed therapy,wherein the mammalian tumor overexpresses HER2, the method comprisingthe step of assaying a sample obtained from the mammalian tumor todetect a pattern of expression, phosphorylation or both expression andphosphorylation of one or a plurality of polypeptides consisting of:

(a) IGFR polypeptide;

(b) EGFR polypeptide;

(c) NDF polypeptide;

(d) phosphorylated S6 ribosomal polypeptide;

(e) phosphorylated AKT polypeptide; and

(f) phosphorylated ERK polypeptide;

wherein the particular combination of polypeptides and pattern ofexpression, phosphorylation or both expression and phosphorylationidentifies mammalian tumors that do not respond to a HER2-directedtherapy.

In certain embodiments, the pattern that identifies a mammalian tumor asnot responding is normal or increased expression of IGFR polypeptide,accompanied by increased phosphorylation of AKT polypeptide, increasedphosphorylation of S6 ribosomal polypeptide, or both in the mammaliantumor as compared to a non-tumor tissue or cell sample. In otherembodiments, the detected pattern is decreased expression of EGFRpolypeptide and increased expression of NDF polypeptide in the mammaliantumor as compared to a non-tumor tissue or cell sample. In furtherembodiments, the detected pattern is decreased expression of EGFRpolypeptide in the mammalian tumor as compared to a non-tumor tissue orcell sample. In other embodiments, the detected pattern is decreasedexpression of NDF polypeptide in the mammalian tumor as compared to anon-tumor tissue or cell sample. In additional embodiments, the detectedpattern is decreased expression of EGFR polypeptide and increasedphosphorylation of ERK polypeptide in the mammalian tumor as compared toa non-tumor tissue or cell sample. In further embodiments, the detectedpattern is normal or increased expression of IGFR polypeptide anddecreased expression of NDF in the mammalian tumor as compared to anon-tumor tissue or cell sample.

In a third aspect, the invention provides methods of selecting a subjectwith cancer for treatment with a molecule targeting HER2, wherein thecancer overexpresses HER2, the methods comprising the steps of:

(a) determining the pattern of expression, phosphorylation or bothexpression and phosphorylation in a cell or tissue sample from thesubject of one or a plurality of polypeptides consisting of:

-   -   (i) IGFR polypeptide;    -   (ii) EGFR polypeptide;    -   (iii) NDF polypeptide;    -   (iv) phosphorylated S6 ribosomal polypeptide;    -   (v) phosphorylated AKT polypeptide; and    -   (vi) phosphorylated ERK polypeptide; and

(b) selecting the subject based on the detected pattern of expression,phosphorylation, or both expression and phosphorylation. The particularcombination of polypeptides and pattern of expression, phosphorylationor both expression and phosphorylation is used to select the subjectswith cancer for treatment with a molecule targeting HER2.

In certain embodiments, the detected pattern for selecting a subject fortreatment with a molecule targeting HER2 is decreased expression of IGFRpolypeptide in the mammalian tumor as compared to a non-tumor tissue orcell sample. In other embodiments, the detected pattern is normal orincreased expression of IGFR polypeptide, accompanied by decreasedphosphorylation of AKT polypeptide, decreased phosphorylation of S6ribosomal polypeptide or both in the mammalian tumor as compared to anon-tumor tissue or cell sample. In further embodiments, the detectedpattern is normal or increased expression of EGFR polypeptide,accompanied by decreased phosphorylation of ERK polypeptide in themammalian tumor as compared to a non-tumor tissue or cell sample. Inadditional embodiments, the detected pattern is decreased expression ofIGFR polypeptide, accompanied by increased phosphorylation of S6ribosomal polypeptide in the mammalian tumor as compared to a non-tumortissue or cell sample. In other embodiments, the detected pattern isdecreased expression of IGFR polypeptide, accompanied by increasedexpression of NDF polypeptide in the mammalian tumor as compared to anon-tumor tissue or cell sample; where further the detected pattern caninclude increased phosphorylation of S6 ribosomal polypeptide.

In a fourth aspect, the invention provides methods of selecting asubject with cancer to not receive treatment with a molecule targetingHER2, wherein the cancer overexpresses HER2, the methods comprising thesteps of:

(a) determining the pattern of expression, phosphorylation or bothexpression and phosphorylation in a cell or tissue sample from thesubject of one or a plurality of polypeptides consisting of:

-   -   (i) IGFR polypeptide;    -   (ii) EGFR polypeptide;    -   (iii) NDF polypeptide;    -   (iv) phosphorylated S6 ribosomal polypeptide;    -   (v) phosphorylated AKT polypeptide; and    -   (vi) phosphorylated ERK polypeptide; and

(b) selecting the subject based on the detected pattern of expression,phosphorylation, or both expression and phosphorylation. The particularcombination of polypeptides and pattern of expression, phosphorylationor both expression and phosphorylation is used to select the subjectswith cancer to not receive treatment with a molecule targeting HER2.

In certain embodiments, the detected pattern for selecting a subject notto receive treatment with a molecule targeting HER2 is normal orincreased expression of IGFR polypeptide, accompanied by increasedphosphorylation of AKT polypeptide, increased phosphorylation of S6ribosomal polypeptide, or both in the mammalian tumor as compared to anon-tumor tissue or cell sample. In other embodiments, the detectedpattern is decreased expression of EGFR polypeptide and increasedexpression of NDF polypeptide in the mammalian tumor as compared to anon-tumor tissue or cell sample. In further embodiments, the detectedpattern is decreased expression of EGFR polypeptide in the mammaliantumor as compared to a non-tumor tissue or cell sample. In otherembodiments, the detected pattern is decreased expression of NDFpolypeptide in the mammalian tumor as compared to a non-tumor tissue orcell sample. In additional embodiments, the detected pattern isdecreased expression of EGFR polypeptide and increased phosphorylationof ERK polypeptide in the mammalian tumor as compared to a non-tumortissue or cell sample. In further embodiments, the detected pattern isnormal or increased expression of IGFR polypeptide and decreasedexpression of NDF in the mammalian tumor as compared to a non-tumortissue or cell sample.

In various aspects of the invention, including those mentioned above,the detection of phosphorylation of AKT polypeptide, phosphorylation ofS6 ribosomal polypeptide, or both can be determined subsequent tocontacting the sample obtained from the mammalian tumor with aHER2-directed therapy. Further, the HER2-directed therapy can be orcomprise rhuMAb HER2 (HERCEPTIN®). In addition, the sample can becontacted with at least one chemotherapeutic agent. Further, thedetected pattern of expression, phosphorylation, or both, of one or aplurality of polypeptides (a) through (f) can be determined using abiodetection reagent. The biodetection reagent can be an antibody or anucleic acid probe. Further, the detected pattern of phosphorylated AKTpolypeptide can be determined using an antibody specific for an epitopecomprising a phosphorylated serine residue at position 473, the detectedpattern of phosphorylated S6 ribosomal polypeptide can be determinedusing an antibody specific for an epitope comprising a phosphorylatedserine residue at position 235, and/or the detected pattern ofphosphorylated ERK polypeptide can be determined using an antibodyspecific for an epitope comprising a phosphorylated threonine residue atposition 202 and a phosphorylated tyrosine residue at position 204.Further, the sample obtained from the mammalian tumor can be aparaffin-embedded biopsy sample. Also, the mammalian tumor can beidentified as overexpressing HER2 using an antibody that binds HER2polypeptide.

In a fifth embodiment, the invention provides kits for characterizing amammalian tumor's responsiveness to a HER2-directed therapy, the kitcomprising:

(a) an antibody that binds IGFR polypeptide,

and one or more of the following:

(b) an antibody that binds phosphorylated AKT polypeptide;

(c) an antibody that binds phosphorylated S6 ribosomal polypeptide;

(d) an antibody that binds EGFR polypeptide;

(e) an antibody that binds HER2 polypeptide;

(f) an antibody that binds NDF polypeptide; and

(g) an antibody that binds phosphorylated ERK polypeptide.

In certain embodiments, the antibody of (b) is immunologically specificfor AKT polypeptide having a phosphorylated serine residue at position473; antibody of (c) is immunologically specific for S6 ribosomalpolypeptide having a phosphorylated serine residue at position 235;and/or the antibody of (f) is immunologically specific for EKTpolypeptide having a phosphorylated threonine residue at position 202and a phosphorylated tyrosine at position 204. In other embodiments, thekit further comprises at least one secondary antibody that binds to anantibody of subpart (a) through (g).

Specific embodiments of the present invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Jan. 12, 2015,and is 10,207 bytes, which is incorporated by reference herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides methods for predicting response in cancersubjects to cancer therapy, including human cancer patients. Inaddition, this invention provides predictive biomarkers to identify thecancer patients for whom the administering a therapeutic agent will bemost effective, including a therapeutic agent for treating breastcancer. Specifically, this invention provides predictive biomarkers forassessing the efficacy of therapeutic agents targeted to HER2/neu,including agents such as HERCEPTIN®.

In contrast to traditional anticancer methods, where chemotherapeuticdrug treatment is undertaken as an adjunct to and after surgicalintervention, neoadjuvant (or primary) chemotherapy consists ofadministering drugs as an initial treatment in certain cancer patients.One advantage of such an approach is that, for primary tumors of morethan 3 cm, it permits the later or concomitant use of conservativesurgical procedures (as opposed to, e.g., radical mastectomy in breastcancer patients) for the majority of patients, due to the tumorshrinking effect of the chemotherapy. Another advantage is that for manycancers, a partial and/or complete response is achieved in abouttwo-thirds of all patients. Finally, because the majority of patientsare responsive after two to three cycles of chemotherapeutic treatment,it is possible to monitor the in vivo efficacy of the chemotherapeuticregimen employed, in order to identify patients whose tumors arenon-responsive to chemotherapeutic treatment. Timely identification ofnon-responsive tumors allows the clinician to limit a cancer patient'sexposure to unnecessary side-effects of treatment and to institutealternative treatments. Unfortunately, methods present in the art,including histological examination, are insufficient for such a timelyand accurate identification. The present invention provides methods fordeveloping more informed and effective regimes of therapy that can beadministered to cancer patients with an increased likelihood of aneffective outcome (i.e., reduction or elimination of the tumor).

A cancer diagnosis, both an initial diagnosis of disease and subsequentmonitoring of the disease course (before, during, or after treatment) isconventionally confirmed through histological examination of cell ortissue samples removed from a patient. Clinical pathologists need to beable to accurately determine whether such samples are benign ormalignant and to classify the aggressiveness of tumor samples deemed tobe malignant, because these determinations often form the basis forselecting a suitable course of patient treatment. Similarly, thepathologist needs to be able to detect the extent to which a cancer hasgrown or gone into remission, particularly as a result of or consequentto treatment, most particularly treatment with chemotherapeutic orbiological agents.

Histological examination traditionally entails tissue-stainingprocedures that permit morphological features of a sample to be readilyobserved under a light microscope. A pathologist, after examining thestained sample, typically makes a qualitative determination of whetherthe tumor sample is malignant. It is difficult, however, to ascertain atumor's aggressiveness merely through histological examination of thesample, because a tumor's aggressiveness is often a result of thebiochemistry of the cells within the tumor, such as protein expressionor suppression and protein phosphorylation, which may or may not bereflected by the morphology of the sample. Therefore, it is important tobe able to assess the biochemistry of the cells within a tumor sample.Further, it is desirable to be able to observe and quantitate both geneexpression and protein phosphorylation of tumor-related genes orproteins, or more specifically cellular components of tumor-relatedsignaling pathways.

Cancer therapy can be based on molecular profiling of tumors rather thansimply their histology or site of the disease. Elucidating thebiological effects of targeted therapies in tumor tissue and correlatingthese effects with clinical response helps identify the predominantgrowth and survival pathways operative in tumors, thereby establishing apattern of likely responders and conversely providing a rationale fordesigning strategies to overcome resistance. Successful diagnostictargeting of a growth factor receptor must determine if tumor growth orsurvival is being driven by the targeted receptor or receptor family, byother receptors not targeted by the therapy, and whether downstreamsignaling suggests that another oncogenic pathway is involved.

For subjects considered for treatment with HERCEPTIN®, it is necessaryto consider additional biomarkers beyond the presence of the targetHER-2/neu, at least because the status of the EGFR and erbB ligands NDFand TGF-α affect HERCEPTIN® therapy response in breast cancer patients.Therefore, considering HER2/neu expression alone does not necessarilypredict overall erbB oncogenic activity or potential response to erbBinhibitors. In addition, previous studies have shown that not all tumorcells respond to inhibition of erbB receptors, despite exhibitingaberrant EGFR and/or HER2/neu expression. Examples include the MKN7 andBT474 erbB receptor-overexpressing carcinoma cell lines: BT474 cellsrespond to HERCEPTIN® but MKN7 cells do not (Motoyama, et al., CancerResearch, 62, 3151-3158 (2002)). In addition, the proliferation blockinduced as a consequence of decreased EGFR or HER2 receptor activity,such as by the activity of an erbB-inhibitor, may be overcome by thepresence of EGF-related ligands such as EGF or NDF/Heregulin (Id). Thisphenomenon can be attenuated using a bispecific erbB-1/erbB-2 inhibitor,thus supporting increased antitumor efficacy through simultaneousinhibition of multiple erbB receptors.

In addition, in many cancers NDF/Heregulin or TGF-α expression producesan autocrine loop of HER-2/EGFR heterodimerization. Downregulation ofHER-2/neu expression is an important way of inhibiting signals generatedby these heterodimers. Downregulation can be accomplished by treatmentwith HERCEPTIN®, which produces receptor endocytosis. Furthermore, highlevels of phosphorylated ERK (or pAKT) can be a negative predictor forpositive treatment outcome, when observed in conjunction with theexpression of EGFR and the presence of NDF, suggesting the existence ofother pathways that might promote proliferation of the tumor cellulargrowth. High pERK is also associated with resistance to HERCEPTIN®through downregulation of p27; this may implicate other signals (such asestrogen receptor's cross activation of the MAPK and AKT pathways) thatmay contribute to high pERK and thus contribute to proliferation of thetumor cells' growth. In addition, phosphorylated AKT has been shown tobe an important part of the response to HERCEPTIN®, as highpAKT-positive patients had poor response to HERCEPTIN®.High-phosphorylated AKT has been shown to be associated with highexpression of insulin like growth factor receptors (IGFR-1) as well asPDGFR and results in resistance to HERCEPTIN®. Interestingly, data fromclinical trials have shown that using a dual inhibitor (i.e., specificfor HER-1/neu and HER-2/neu) has clinical efficacy in patients whentreatment induced downregulation of pERK and pAKT, but not in patientsin which pERK and pAKT levels didn't diminish after treatment. Thus, inthose patients who overexpressed HER-1 and HER-2, as well as pERK andpAKT, antitumor activity was dependent on HER-1 and HER-2 receptoractivation and a clinical response was observed. In contrast, inpatients for whom pERK and pAKT activity remained high after treatmentwith a dual inhibitor, clinical response didn't occur. Combinationtherapies can have clinical significance. A combination of theerbB-1-directed monoclonal antibodies mAb 225 and mAb 4D5 inhibitedproliferation of an ovarian tumor cell line more strongly than eithermAb alone (Ye et al., 1999, Oncogene 18: 731-8). In addition toerbB-targeted mAbs, a number of different erbB-1/erbB-2-bispecificinhibitors, also referred to as dual EGFR/erbB-2 kinase inhibitors, havebeen described recently, such as GW572016 and PKI166, that are currentlyin clinical trials (Motoyama et al., 2002, Cancer Research 62:3151-3158). Therefore, response to HERCEPTIN® is affected by theexpression of multiple erbB receptors and their ligands in tumors.

Thus, HER-2/neu overexpression alone is not the only predictor ofresponse to molecules such as HERCEPTIN®. HER-2/neu is an orphan,ligandless receptor in need of its partners EGFR (HER-1) and HER-3 inorder to exert its activity. HER-1 and HER-3 heterodimerization withHER-2 is enhanced by the presence of EGF or NDF (Tzahar et al., 1996,EMBO J. 15: 254-64, Graus-Porta, 1997, EMBO J., 16 1647-55), and thusHER-2 activity is dependent on HER-1 or HER-3. Other receptors may alsotransactivate the erbB receptors. These receptors may be mediatingtumorigenesis through signaling to downstream proliferative and survivalpathways. For example, the IGFR receptor may mediate patient response tobreast cancer therapies targeting HER2/neu. High IGFR expressioncombined with high S6 ribosomal protein phosphorylation correlates withpoor patient response regardless of erb-B expression, indicating thatIGFR acts directly to activate signaling downstream of erb-B receptorsrather than through transactivation of erb-B receptors. Cell linestudies also have suggested a role for IGFR in patient response.HERCEPTIN® resistance has been suggested to occur through activation ofIGFR (Lu et al., 2001, J. National Cancer Institute 93: 1852). Inaddition, co-targeting IGFR as well as HER2/neu has been shown toproduce synergistic inhibition of growth in breast cancer cells(Camirand et al., 2002, Med Sci Monit. 8: (12): BR521-6). Therefore,analysis of IGFR expression and downstream signaling can be critical foran accurate assessment of potential HERCEPTIN® response in breast cancerpatients.

Thus, there is no one marker of downstream signaling protein activationthat would integrate multiple upstream signals and predict response.However, analysis of p-ERK and p-AKT has been found to be predictive inpatients over-expressing EGFR. Therefore, in the presence of active erbBreceptors, high ERK and AKT signaling indicates that HERCEPTIN® therapyis less likely to be effective. AKT activation has been shown to resultin HERCEPTIN® resistance in breast cancer cell lines (Yakes, et al.,2002, Cancer Res. 62: 4132-41; Clark et al., 2002, Mol. Cancer Ther. 1:707-17). In addition, analysis of S6 ribosomal protein phosphorylationgreatly increased the predictive power of IGFR expression. In patientswith high S6 phosphorylation, positive response ranged from 8% to 67%based upon IGFR expression. Approximately 30% of patients with low S6phosphorylation responded, regardless of IGFR expression. These resultswere also reflected in an analysis of clinical samples, in which onlythose patients that lacked active IGFR signaling responded to HERCEPTIN®therapy. IGF signaling in breast cancer occurs through AKT activation(Oh et al., 2002, Neoplasia 4: 204-17; Dufourny et al., 1997, J BiolChem. 272: 31163-71), which leads to S6 ribosomal proteinphosphorylation. Hence, S6 phosphorylation can be indicative of activeIGF signaling in those tumors over-expressing IGFR.

Analysis of down-stream signaling and patient response is complicatedwhen chemotherapy and radiotherapy therapy is included in addition toHERCEPTIN® treatment. AKT and MAP kinase pathway activation, forexample, are known to play a role in response to DNA-damaging agents(Clark et al., 2002, Mol. Cancer Ther. 1: 707-17; Bacus et al., 2001,Oncogene 20: 147-155). Consideration of downstream signaling in patientsundergoing a combination of therapies provides additional predictiveinformation not obtained solely from analysis of receptor or ligandexpression levels. Analysis of patients treated with HERCEPTIN® as asingle agent therapy can be used to determine which of the identifiedbiomarkers mediated the response to Herceptin® as opposed to thebiomarkers that mediate the response to the other therapies.Nonetheless, the identified biomarkers are useful, among other things,for designing diagnostics for breast cancer patients undergoing thecommon HERCEPTIN® combination therapies.

Further, up-regulation of the AKT/mTOR pathway by Heregulin/NDF is animportant predictor for response. pAKT has been associated with highlevels of Cyclin E and low levels of the cyclin inhibitor p27.

Before administration of HER2-targeted therapies, a panel of diagnosticsof each tumor is used according to the methods of this invention to findthe best candidate for each therapy. According to the methods of thisinvention, treatment by a HER2-targeted therapy, such as HERCEPTIN®, iseffective when a patient's tumor growth depends on a cellular pathwaysuch as AKT/mTOR that is driven by the erbB receptors and not by othertyrosine kinases, such as Insulin-like Growth Factor Receptors (IGFR).When high levels of activation of these downstream signals occurindependent of erbB receptors, HERCEPTIN® treatment is not effective.Use of the methods of this invention permits a clinician to choose amore effective combination of targeted therapies for cancer patients.

The HER2-directed therapies of the present invention can include, forexample, rhuMAb HER2, otherwise known as HERCEPTIN®. The samplesobtained from the mammalian tumor can be contacted with at least onechemotherapeutic agent, for example cisplatin, doxorubicin, or taxol.

Automated (computer-aided) image analysis systems known in the art canaugment visual examination of tumor samples. In a representativeembodiment, the cell or tissue sample is exposed to detectably-labeledreagents specific for a particular biological marker, and the magnifiedimage of the cell is then processed by a computer that receives theimage from a charge-coupled device (CCD) or camera such as a televisioncamera. Such a system can be used, for example, to detect and measureexpression and activation levels of EGFR, HER2, HER3, pERK, NDF, TGF-α,IGFR, pS6, and pAKT in a sample, or any additional diagnosticbiomarkers. Thus, the methods of the invention provide more accuratecancer diagnosis and better characterization of gene expression inhistologically identified cancer cells, most particularly with regard toexpression of tumor marker genes or genes known to be expressed inparticular cancer types and subtypes (e.g., having different degrees ofmalignancy). This information permits a more informed and effectiveregimen of therapy to be administered, because drugs with clinicalefficacy for certain tumor types or subtypes can be administered topatients whose cells are so identified.

Another drawback of conventional anticancer therapies is that theefficacy of specific chemotherapeutic agents in treating a particularcancer in an individual human patient is unpredictable. In view of thisunpredictability, the art is unable to determine, prior to startingtherapy, whether one or more selected agents would be active asanti-tumor agents or to render an accurate prognosis of course oftreatment in an individual patient. This is especially important becausea particular clinical cancer may present the clinician with a choice oftreatment regimens, without any current way of assessing which regimenwill be most efficacious for a particular individual. It is an advantageof the methods of this invention that they are able to better assess theexpected efficacy of a proposed therapeutic agent (or combination ofagents) in an individual patient. The claimed methods are advantageousfor the additional reasons that they are both time- and cost-effectivein assessing the efficacy of chemotherapeutic regimens and are minimallytraumatic to cancer patients.

Methods of this invention can be used to identify a mammalian tumor thatresponds to HER-2 directed therapies. Further, methods of this inventioncan be used to select a subject with cancer for treatment with amolecule targeting HER. Moreover, methods of this invention can be usedto identify a mammalian tumor that does not respond to HER-2 directedtherapies. Further, methods of this invention can be used to select asubject with cancer to not receive treatment with a molecule targetingHER2.

Patterns of expression and phosphorylation of polypeptides are detectedand quantified using methods of the present invention. Moreparticularly, patterns of expression and phosphorylation of polypeptidesthat are cellular components of a tumor-related signaling pathway aredetected and quantified using methods of the present invention. Forexample, the patterns of expression and phosphorylation of polypeptidescan be detected using biodetection reagents specific for thepolypeptides. For example, the biodetection reagents can be antibodies.Alternatively, the biodetection reagents can be nucleic acid probes. Anucleic acid probe is defined to be a collection of one or more nucleicacid fragments whose hybridization to a sample can be detected. Theprobe may be unlabeled or labeled so that its binding to the target orsample can be detected. The probe is produced from a source of nucleicacids from one or more particular (preselected) portions of the genome,e.g., one or more clones, an isolated whole chromosome or chromosomefragment, or a collection of polymerase chain reaction (PCR)amplification products. The nucleic acid probe may also be isolatednucleic acids immobilized on a solid surface (e.g., nitrocellulose,glass, quartz, fused silica slides), as in an array. The probe may be amember of an array of nucleic acids as described, for instance, in WO96/17958. Techniques capable of producing high density arrays can alsobe used for this purpose (see, e.g., Fodor (1991) Science 767-773;Johnston (1998) Curr Biol. 8: R171-R174; Schummer (1997) Biotechniques23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No.5,143,854). One of skill will recognize that the precise sequence of theparticular probes can be modified to a certain degree to produce probesthat are “substantially identical,” but retain the ability tospecifically bind to (i.e., hybridize specifically to) the same targetsor samples as the probe from which they were derived. The term “nucleicacid” refers to a deoxyribonucleotide or ribonucleotide in eithersingle- or double-stranded form. The term encompasses nucleic acids,i.e., oligonucleotides, containing known analogues of naturalnucleotides that have similar or improved binding properties, for thepurposes desired, as the reference nucleic acid. The term also includesnucleic acids which are metabolized in a manner similar to naturallyoccurring nucleotides or at rates that are improved for the purposesdesired. The term also encompasses nucleic-acid-like structures withsynthetic backbones. One of skill in the art would recognize how to usea nucleic acid probe for screening of cancer cells in a sample byreference, for example, to U.S. Pat. No. 6,326,148, directed toscreening of colon carcinoma cells.

Polypeptides associated with breast cancer can be quantified by imageanalysis using a suitable primary antibody against biomarkers, includingbut not limited to EGFR, HER-2, HER-3, IGFR, NDF, TGF-α, p-ERK, pS6, orp-AKT, detected directly or using an appropriate secondary antibody(such as rabbit anti-mouse IgG when using mouse primary antibodies)and/or a tertiary avidin (or Streptavidin) biotin complex (“ABC”).

Examples of reagents useful in the practice of the methods of theinvention as exemplified herein include antibodies specific forHER2/neu, including but not limited to the mouse monoclonal antibodyCB11, obtained from Ventana Medical Systems, Inc. (VMSI, Tucson, Ariz.).In addition, reagents useful in the practice of the methods of theinvention include antibodies specific for phosphorylated AKT, includingbut not limited to antibodies specific for a phosphorylated serineresidue of position 473, wherein the sequence of AKT is represented bySEQ ID NO:1 (Table 8). Further, reagents useful in the practice of themethods of the invention include antibodies specific for phosphorylatedS6, including but not limited to antibodies specific for aphosphorylated serine residue of position 235, wherein the sequence ofS6 is represented by SEQ ID NO:2 (Table 8). Also, reagents useful in thepractice of the methods of the invention include antibodies specific forphosphorylated ERK, including but not limited to antibodies specific fora phosphorylated threonine residue at position 202 and a phosphorylatedtyrosine residue of position 204, wherein the sequence of ERK isrepresented by SEQ ID NO:3 (Table 8).

Further, the pattern of expression, phosphorylation, or both expressionand phosphorylation of the predictive polypeptides can be compared to anon-tumor tissue or cell sample. The non-tumor tissue or cell sample canbe obtained from a non-tumor tissue or cell sample from the sameindividual, or alternatively, a non-tumor tissue or cell sample from adifferent individual. A detected pattern for a polypeptide is referredto as decreased in the mammalian tumor, tissue, or cell sample, if thereis less polypeptide detected as compared to the a non-tumor tissue orcell sample. A detected pattern for a polypeptide is referred to asincreased in the mammalian tumor, tissue, or cell sample, if there ismore polypeptide detected as compared to the a non-tumor tissue or cellsample. A detected pattern for a polypeptide is referred to as normal inthe mammalian tumor, tissue, or cell sample, if there is the same, orapproximately the same, polypeptide detected as compared to the anon-tumor tissue or cell sample.

The methods of this invention for identifying mammalian tumors thatrespond, or that do not respond, to a HER2-directed therapy comprise thestep of assaying a sample obtained from the mammalian tumor to detect apattern of expression, phosphorylation or both of one or a plurality ofpolypeptides consisting of: (a) IGFR polypeptide; (b) EGFR polypeptide;(c) NDF polypeptide; (d) phosphorylated S6 ribosomal polypeptide; (e)phosphorylated AKT polypeptide; (f) phosphorylated EKT polypeptide. Thecombination of polypeptides and pattern of expression, phosphorylation,or both expression and phosphorylation identifies mammalian tumors thatrespond, or that do not respond, to a HER2-directed therapy. The methodscan include the detection of a pattern of expression, phosphorylation orboth of one, two, three, four, five, or all six of these polypeptides.Further, the methods can, but need not, include other steps, includingsteps such as the detection of a pattern of expression, phosphorylationor both of different polypeptides.

The methods of this invention for selecting a subject with cancer fortreatment, or to not receive treatment, with a molecule targeting HER2,such as, but not limited to treatment with HERCEPTIN®, comprise the stepof determining the pattern of expression, phosphorylation or both in acell or tissue sample from the subject of one or a plurality ofpolypeptides consisting of: (a) IGFR polypeptide; (b) EGFR polypeptide;(c) NDF polypeptide; (d) phosphorylated S6 ribosomal polypeptide; (e)phosphorylated AKT polypeptide; (f) phosphorylated EKT polypeptide. Thecombination of polypeptides and pattern of expression, phosphorylation,or both expression and phosphorylation is used to select a subject withcancer for treatment, or to not receive treatment, with a moleculetargeting HER2. The methods can include the detection of a pattern ofexpression, phosphorylation or both of one, two, three, four, five, orall six of these polypeptides. Further, the methods can, but need not,include other steps, including steps such as the detection of a patternof expression, phosphorylation or both of different polypeptides.

For example, the pattern that identifies a mammalian tumor as respondingor that can be used to select a subject with cancer for treatment with amolecule targeted to HER2 is decreased expression of IGFR polypeptide ascompared to a non-tumor tissue or cell sample. Alternatively, thedetected pattern is normal or increased expression of IGFR polypeptide,accompanied by decreased phosphorylation of AKT polypeptide, decreasedphosphorylation of S6 ribosomal polypeptide or both as compared to anon-tumor tissue or cell sample. Another potential detected pattern isnormal or increased expression of EGFR polypeptide, accompanied bydecreased phosphorylation of ERK polypeptide as compared to a non-tumortissue or cell sample. Further detected patterns include decreasedexpression of IGFR polypeptide, accompanied by increased phosphorylationof S6 ribosomal polypeptide as compared to a non-tumor tissue or cellsample. In other embodiments, the detected pattern is decreasedexpression of IGFR polypeptide, accompanied by increased expression ofNDF polypeptide in the mammalian tumor as compared to a non-tumor tissueor cell sample; where further the detected pattern can include increasedphosphorylation of S6 ribosomal polypeptide. These identified patternsare understood to be non-limiting.

For example, the pattern that identifies a mammalian tumor as notresponding or that can be used to select a subject with cancer to notreceive treatment with a molecule targeted to HER2 is normal orincreased expression of IGFR polypeptide, accompanied by increasedphosphorylation of AKT polypeptide, increased phosphorylation of S6ribosomal polypeptide, or both as compared to a non-tumor tissue or cellsample. Alternatively, the detected pattern is decreased expression ofEGFR polypeptide and increased expression of NDF polypeptide as comparedto a non-tumor tissue or cell sample. Or, the detected pattern isdecreased expression of EGFR polypeptide as compared to a non-tumortissue or cell sample. Further, the detected pattern is decreasedexpression of NDF polypeptide as compared to a non-tumor tissue or cellsample. Or, the detected pattern is decreased expression of EGFRpolypeptide and increased phosphorylation of ERK polypeptide as comparedto a non-tumor tissue or cell sample. Further, the detected pattern isnormal or increased expression of IGFR polypeptide and decreasedexpression of NDF as compared to a non-tumor tissue or cell sample.These identified patterns are understood to be non-limiting.

In practicing the methods of this invention, staining procedures can becarried out by a person, such as a technician in the laboratory.Alternatively, the staining procedures can be carried out usingautomated systems. In either case, staining procedures for use accordingto the methods of this invention are performed according to standardtechniques and protocols well-established in the art.

By “cell or tissue sample” is meant biological samples comprising cells,most preferably tumor cells, that are isolated from body samples, suchas, but not limited to, smears, sputum, biopsies, secretions,cerebrospinal fluid, bile, blood, lymph fluid, urine and faeces, ortissue which has been removed from organs, such as breast, lung,intestine, skin, cervix, prostate, and stomach. For example, a tissuesamples can comprise a region of functionally related cells or adjacentcells.

The amount of target protein is advantageously quantified by measuringthe average optical density of the stained antigens. Concomitantly, theproportion or percentage of total tissue area stained can be readilycalculated, for example as the area stained above a control level (suchas an antibody threshold level) in the second image. Followingvisualization of nuclei containing biomarkers, the percentage or amountof such cells in tissue derived from patients after treatment arecompared to the percentage or amount of such cells in untreated tissue.For purposes of the invention, “determining” a pattern of expression,phosphorylation, or both expression and phosphorylation of polypeptidesis understood broadly to mean merely obtaining the information on suchpolypeptide(s), either through direct examination or indirectly from,for example, a contract diagnostic service.

Breast cancer tissue sections taken from patients treated withHERCEPTIN® and chemotherapy are analyzed, according to the methods ofthis invention by immunohistochemistry for expression, phosphorylation,or expression and phosphorylation of erb-B ligands, receptors,downstream signaling proteins or any positive treatment responsepredictive combination thereof. These measurements can be accomplished,for example, by using tissue microarrays. Tissue microarrays areadvantageously used in the methods of the invention, being awell-validated method to rapidly screen multiple tissue samples underuniform staining and scoring conditions (Hoos et al., 2001, Am J Pathol.158: 1245-51). Scoring of the stained arrays can be accomplished by anautomated system that accurately quantifies the staining observed. Theresults of this analysis identify biomarkers that best predict patientoutcome following treatment, such as HERCEPTIN® therapies. Patient“probability of response” ranging from 0 to 100 percent can be predictedbased upon the expression, phosphorylation or both of a small set ofligands, receptors, signaling proteins or predictive combinationthereof. Additional samples from breast cancer patients can be analyzed,either as an alternative to or in addition to tissue microarray results.For example, analysis of samples from breast cancer patients can confirmthe conclusions from the tissue arrays, if the patient's responsescorrelate with a specific pattern of receptor expression and/ordownstream signaling.

The invention provides, in part, kits for carrying out the methods ofthe invention. For example, the method provides kits for characterizinga mammalian tumor's responsiveness to a HER2-directed therapy comprisingan antibody that binds IGFR polypeptide, and one or more of thefollowing: an antibody that binds phosphorylated AKT polypeptide; anantibody that binds phosphorylated S6 ribosomal polypeptide; an antibodythat binds EGFR polypeptide; an antibody that binds HER2 polypeptide; anantibody that binds NDF polypeptide; and an antibody that bindsphosphorylated ERK polypeptide. In addition to an antibody that bindsIGFR polypeptide, the kit can include one, two, three, four, or all fiveof the following: an antibody that binds phosphorylated AKT polypeptide;an antibody that binds phosphorylated S6 ribosomal polypeptide; anantibody that binds EGFR polypeptide; an antibody that binds HER2polypeptide; an antibody that binds NDF polypeptide; and an antibodythat binds phosphorylated ERK polypeptide. Further, the kit can includeadditional components other than the above-identified antibodies,including but not limited to additional antibodies. Such kits may beused, for example, by a clinician or physician as an aid to selecting anappropriate therapy for a particular patient, for example, a breastcancer patient under consideration for HER2-directed therapy.

Particularly useful embodiments of the present invention and theadvantages thereof can be understood by referring to Examples 1-5. TheseExamples are illustrative of specific embodiments of the invention, andvarious uses thereof. They are set forth for explanatory purposes only,and are not to be taken as limiting the invention.

Example 1 Staining Procedure for Biomarkers

Human tumor tissue sections were stained for predictive biomarkersaccording to the methods of the invention as follows. 10% NeutralBuffered Formalin Paraffin blocks were sectioned at 4 microns and thesections placed onto coated slides. EGFR and HER2 immunostaining wasperformed by using the pre-diluted EGFR and HER2 antibodies from VentanaMedical Instruments, Inc. (VMSI, Tucson, Ariz.). HER3, Heregulin (NDF),and IGFR antibodies were obtained from NeoMarkers (Fremont, Calif.).TGF-α antibodies were obtained from Oncogene Sciences (San Diego,Calif.). EGFR, HER2/neu, HER3, IGFR, Heregulin, and TGF-α wereimmunostained using the “BenchMark” (VMSI) with I-VIEW (VMSI) detectionchemistry. Antibodies specific for p-ERK (1:100), p-AKT (1:75), andphospho-S6 ribosomal protein were obtained from Cell SignalingTechnology (Beverly, Mass.), and immunostained using a labeledstreptavidin peroxidase technique (Vector Elite ABC Kit, Burlingame,Calif.). Prior to staining, slides for p-S6 ribosomal protein, p-ERK andp-AKT were antigen retrieved using 0.1 M citrate buffer, pH 6.0 in the“decloaker” (Biocare Corp.) and the sections incubated overnight withthe primary antibodies at 4° C. The next day, the slides were placedonto the Autostainer (Dako Corp.) and the “LSAB2” kit (Dako) wasemployed as the detection chemistry. DAB (Dako) was used as thechromogen. After immunostaining, all slides were counterstained manuallywith 4% ethyl green (Sigma).

Example 2 Procedure for Western Blot Analysis

Western blot analysis for detecting expression of predictive markers wasperformed as follows. Cells were lysed in ice-cold buffer (50 mMTris-HCl (pH 7.5), 137 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.2% TritonX-100, 10% glycerol, 0.1 mM sodium orthovanadate, 10 mM sodiumpyrophosphate, 20 mM β-glycerophosphate, 50 mM NaF, 1 mMphenylmethylsulfonyl fluoride, 2 μM leupeptin, and 2 μg/ml aprotinin).Protein concentration was determined with a BioRad Protein Assay Kit(BioRad Laboratories, Hercules, Calif.). Equal amounts of protein,typically 15 μg protein per lane, were separated by gel electrophoresis,for example using pre-cast 4-12% Bis-Tris NuPage gradient gels(Invitrogen) or 7.5% or 4-15% gradient SDS-PAGE under reducingconditions, and transferred to membranes, such as HyBond-Cnitrocellulose (Amersham Life Science) or Immobilon-P membranes.Membranes were blocked and then incubated with primary antibodies, forexample antibodies against p-AKT and p-ERK (Cell Signaling Technology).Antibody incubation was performed overnight at 4° C. in Tris-bufferedsaline containing 3% bovine serum albumin/0.1% Tween 20. Signal wasdetected by chemiluminescence (PerkinElmer Life Sciences), or using aSuperSignal West Femto Maximum sensitivity substrate kit from Pierce(Rockford, Ill.) as described (Xia et al., 2002, Oncogene 21:6255-6263).

Example 3 Procedure for Immunohistochemistry

Immunohistochemistry for detecting and measuring predictive biomarkerexpression, activation or both was performed as follows. HER2/neu, EGFR,HER3, IGFR, TGF-α, Heregulin (NDF), p-ERK, p-AKT, and p-S6 ribosomalprotein or phosphorylation levels were quantified using alkalinephosphatase or peroxidase techniques and microscope-based image analysisof immunohistochemically stained slides (as described in Bacus et al.,1997, Analyt. Quant. Cytol. Histol. 19: 316-328). Quantification was bymeans of a CAS 200 image analyzer, as previously described (Bacus &Ruby, 1993, Pathol Annu, 28: 179-204). For the purpose of the analysis,tumors were classified as negative or positive for each antibody basedupon the level of staining. Statistical analysis was performed usingSystat to quantify frequencies and calculate Pearson Chi-squared testsof significance for interactions between variables. In all cases, the pvalue refers to the significance of the deviation of the distribution ofsamples from what would be expected based upon the overall populationdistribution. Comparisons were performed only on samples for which allrelevant data were available. As a result, the number of patientsincluded in most comparisons was slightly less than the total number ofavailable samples.

Quantitative immunohistochemistry (IHC) was performed as previouslydescribed (Bacus et al., 1997, Analyt. Quant. Cytol. Histol. 19:316-328). EGFR, and erbB-2 (HER2) immunostaining was performed usingpre-diluted EGFR, and erbB-2 (HER2) antibodies from Ventana MedicalSystems, Inc. (VMSI, Tucson, Ariz.) on the VMSI automated “BenchMark”staining module as described. The VMSI “I-View” detection kit was usedfor all three of the VMSI pre-diluted primary antibodies according tothe manufacturer's instructions. Antibodies to erbB-3 (1:10), Heregulin(1:25), and TGF-α (1:20), were also used for immunostaining using the“BenchMark” with I-VIEW detection chemistry. Antibodies to Phospho-Erk(1:100) and p-AKT (1:75) were used for immunostaining using a labeledstreptavidin peroxidase technique as described by the manufacturer.Phospho-Erk and p-AKT slides were antigen retrieved as described byBacus et al. (1997, Analyt. Quant. Cytol. Histol. 19: 316-328). Slideswere placed onto the Autostainer (Dako Corp.) and the “LSAB2” kit (Dako)employed as the detection chemistry. After staining, all slides werecounterstained manually with 4% ethyl green (Sigma). Investigatorspreparing and analyzing tissue sections were blinded to both patienttumor type and response to therapy.

For IHC, antibodies to EGFR and erbB-2 were from Ventana MedicalSystems, Inc. (VMSI) (Tucson, Ariz.); anti-p-AKT (Ser 437) and p-Erk1/2were from Cell Signaling Technology, Inc. (Beverly, Mass.); antibodiesto TGFα, erbB3, heregulin, and IGFR-1 were from NeoMarkers.

Example 4 Analysis of Breast Cancer Tissue Microarrays

Tissue microarrays derived from 250 breast cancer patients who receivedconventional chemotherapy together with HERCEPTIN® were obtained fromClinomics Biosciences (Pittsfield, Mass.). The histology of the tumorsvaried, with infiltrating ductal carcinoma being the most common. Allpatients had received post-surgical radiotherapy. The tissue samples inthe array were taken before treatment. HER2/neu expression had beendetermined by using the HercepTest system (DAKO, Carpinteria, Calif.) onthe original biopsies for all patients. Patient response was based uponthe case histories at last follow-up as decided by an independentpathologist provided by Clinomics.

Demographics of these patients are reported in Table 1. The greatmajority of patients had infiltrating ductal carcinomas and receivedanthracycline plus cyclophosphamide. Fifty-seven of the patients hadmetastatic diseases. All patients had received a 4 mg/kg HERCEPTIN®loading dosage and a 2 mg/kg weekly maintenance dosage.

From the original tissue arrays of 250 patients, seventy-five sampleswere not used in the analysis because of the lack of clinical data orbecause the sections did not contain useable tumor tissue. Overall, 15%of the remaining patients were disease free or had stable disease aftertherapy, while 85% relapsed. Of these remaining one hundred seventy-fivepatients, twenty-eight samples lacked HercepTest results and weretherefore also excluded from further analysis. Of the samples for whichHercepTest results were obtained, seventy-seven had a HercepTest scoreof +3, and seventy had +2 or less staining intensity (Table 1).

The HercepTest staining scores were confirmed by analyzing HER2/neuexpression levels using microarrays (data not shown). HER2/neuexpression strongly correlated with patient response; 100% of the 0 or+1 HER2/neu patients relapsed while only 77% of the +3 patientsrelapsed. This response rate is similar to what has been reportedpreviously (see Baselga, 2002, Annals of Oncology 13: 8-9). Based onthese results, further analysis of biomarkers concentrated on patientsthat expressed HER2 at the highest (+3) level. Of the samples that hadthe highest HercepTest scores (+3), seventy-four were taken from theprimary tumor, two from lymph nodes, and one from an adrenal metastasis.

The seventy-seven patients who overexpressed HER2/neu (+3 HercepTeststaining score) were analyzed for expression levels of EGFR, HER3, IGFR,and NDF/Heregulin, and TGF-α, as well as activated downstream signalsp-ERK and p-AKT (phosphorylated forms of ERK and AKT) and the downstreamsignal of mTOR, p-S6 (phosphorylated S6 ribosomal protein). The analysisof receptor kinases revealed that, similar to HER2/neu, EGFR expressionalso significantly correlated with patient response (Table 2). Among theHERCEPTIN®-treated patients that over-expressed HER-2/neu, 30% ofEGFR-positive patients had stable disease or were disease free, whileonly 9% of EGFR-negative patients did not progress. Among theseventy-seven +3 HER2/neu patients, seventy of them expressed HER3;however, HER3 expression did not significantly correlate with patientresponse (although the low number of HER3-negative patients limits thiscomparison in the data set). The growth-factor receptor HER3 is thoughtto play an important role in downstream erbB signaling because it has aPI3-Kinase docking site and forms active heterodimers with the othererbB receptors. The expression of other growth factor receptors may alsomediate patient response, either through direct stimulation ordownstream pathways or through transactivation of the erbB receptors.

Expression of erbB ligands, including NDF and TGF-α, also varied amongpatients (see Table 3). Approximately 70% of the patients expressed highlevels of NDF, while approximately 57% expressed high levels of TGF-α. Asignificant correlation was observed between NDF levels and patientresponse. A very high proportion of HER2/neu overexpressing patients whowere NDF-negative relapsed (91%), whereas only 62% of NDF-positivepatients who overexpressed HER2/neu relapsed. In contrast, no predictiverelationship was observed between TGF-α levels alone and patientresponse (see Table 3). The combination of TGF-α or NDF expression andEGFR over-expression, however, did positively correlate with patientresponse in patients overexpressing HER2/neu (p=0.02 and p=0.03respectively) (data not shown).

The activation of heterodimers of HER2 with HER3 or EGFR results inactivation of the MAPK and PI3K/AKT pathways. The MAPK pathway wasmeasured by analyzing the level of activation or phosphorylation of ERK(pERK). Analysis and comparison of the levels of activated ERK alone,among patients that overexpressed HER2/neu and who either had stabledisease or who relapsed, failed to demonstrate any dramatic effect ofelevated pERK levels as a factor for patient response (see Table 4).Similarly, based on this analysis, AKT activation (p-AKT) alone does notappear to be a predictive marker for response among HER2-positivepatients treated with HERCEPTIN® (see Table 4). Also, analysis of S6ribosomal protein phosphorylation, which integrates multiple signalsthrough mTOR and p70 S6 kinase, did not significantly correlate withpatient response for patients that overexpressed HER2/neu (see Table 4).If consideration of pERK and pAKT expression is limited to thosepatients that expressed EGFR and HER2/neu, however, low expression ofeither of these signaling molecules was a significant predictor ofpositive response to HERCEPTIN® (Table 5).

To increase the predictive power of the analysis, consideration of twoor more of the biomarkers were combined in a multivariate analysis tocharacterize the tumor. In this analysis, the observation of thecombination of HER2/neu and EGFR expression with ERK activationsignificantly predicted response (see Table 5). For example, patientswith low EGFR expression and high ERK phosphorylation failed to respond(0% response), whereas patients with high EGFR expression and low ERKphosphorylation had a high response rate (42% response). Similarly, thecombination of high EGFR and HER2/neu with high NDF expression or acombination of high EGFR and HER2/neu with high TGF-α expressionpredicted a better response compared to patients that had low expressionof EGFR and the NDF ligand (data not shown). This comparison was oftendramatic. For example, while 39% of the patients with high EGFR,HER2/neu, and NDF expression responded to therapy, none of the patientswith high HER2/neu expression but low EGFR and NDF expression responded(data not shown).

The combination of high HER2/neu expression, low IGFR expression, andhigh S6 ribosomal protein phosphorylation gave high patient response(67%, Table 5). This is in contrast to patients with high HER2/neu andIGFR expression and high S6 ribosomal protein phosphorylation, a highpercentage that did not respond to therapy. The best combination ofmarkers for predicting whether patients that overexpressed HER2/neuwould respond to HERCEPTIN® therapy were high NDF expression, low IGFRexpression, and high S6 phosphorylation (Table 6). In contrast, none ofthe patients that overexpressed HER2/neu and had low NDF expression andhigh IGFR expression responded to therapy, regardless of S6 status(Table 6). However, these results were obtained using a small samplepopulation of these patients. In patients with high NDF, HER2/neu, andEGFR expression levels, phosphorylation of ERK correlated with adifference in response from 28% (high p-ERK) to 54% (low p-ERK) (Table6). Similarly, those patients with low levels of p-AKT with any othercombination of biomarkers that include the expression of HER2/neu andNDF, did better than those that over-express this protein (results notshown). Taken together, this data shows that HER2/neu together with itsligand and other erbB receptors and ligands, as well as other growthfactor receptors play a role in HERCEPTIN® response. Importantly,analysis of a select combination of these proteins correlated withresponse rates that varied from 0 to 100%.

Example 5 Analysis of Breast Cancer Samples

Samples from seven breast cancer patients were obtained from YaleUniversity. The clinical history of these seven patients varied, withsome given HERCEPTIN® in combination with chemotherapy as a first linetherapy while others were given HERCEPTIN® as an adjuvant therapy. Theseseven samples were analyzed for receptor, ligand, and signaling proteinexpression or phosphorylation, and the results compared to the resultswith the tissue microarray analysis.

All seven patients over-expressed HER2/neu, as determined at the time ofanalysis with the other antibodies immunologically specific fornon-HER2/neu polypeptides. The case histories of the patients varied.For example, patient #1 was given HERCEPTIN® plus docetaxel afterrelapsing with metastatic disease four years after initial presentation.This patient had stable disease for more than a year after commencingcombination therapy. Patient #7 was given HERCEPTIN® plus vinorelbinefollowing the discovery of a solitary metastasis seven months afterinitial radiotherapy. After eight weeks of combination therapy there wasprogression of disease. Of the seven patients, three showed response toHERCEPTIN® while the other four failed to respond (Table 7). One of theresponders did not express IGFR but did express EGFR and showed positivedownstream signaling. The other one of these responders expressed IGFRand EGFR but did not show active downstream signaling in S6 or ERK. Allof the non-responders expressed IGFR and had positive S6phosphorylation. Two of the non-responders also expressed EGFR. Theseresults are consistent with the results obtained from the microarrayanalysis. Patients with active IGFR receptors as demonstrated by IGFRexpression plus S6 phosphorylation are unlikely to respond toHERCEPTIN®, while patients that lack IGFR or have IGFR but no downstreamsignaling are more likely to respond.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

TABLE 1 Demographics number of disease-free or patients stable diseaserelapse all patients included in study 175 15% 85% Histologyinfiltrating ductal carcinoma 109 17% 83% lobular carcinoma 7 43% 57%medullary carcinoma 3 33% 67% metastatic breast carcinoma 19 5% 95%papillary carcinoma 3 0% 100% scirrhous carcinoma 3 100% 0% tubularcarcinoma 3 0% 100% treatment following surgery (followed byHerceptin ®) Doxorubicin 44 2% 98% anthracycline plus cyclophosphamide100 23% 77% Paclitaxel 3 100% 0% HER2/neu expression tumor 0 or 1 17 0%100% 2 53 8% 92% 3 77 30% 70%Demographics of breast cancer patient samples.

TABLE 2 % % patient group n responders relapse P value EGFR positive 4330% 70% 0.002 EGFR negative 23 9% 91% HER3 positive 70 29% 71% 0.43 HER3negative 7 43% 57% IGFR positive 33 24% 76% 0.16 IGFR negative 35 40%60%Receptor tyrosine kinase expression versus patient response. Analysis ontissue array samples for which clinical and Herceptest data wasavailable and who over-expressed HER2/neu.

TABLE 3 % % patient group n responders relapse P value NDF positive 5539% 62% 0.01 NDF negative 22 9% 91% TGF-α positive 38 34% 66% 0.56 TGF-α29 28% 72% negativeReceptor tyrosine kinase ligand expression versus patient responsefollowing therapy. Analysis on tissue array samples for which clinicaland Herceptest data was available and who over-expressed HER2/neu.

TABLE 4 patient group n % responders % relapse P value p-ERK positive 3625% 75% 0.43 p-ERK negative 39 33% 67% p-AKT positive 24 25% 75% 0.53p-AKT negative 53 32% 68% p-S6 positive 27 33% 67% 0.74 p-S6 negative 4430% 70%Downstream protein activation versus patient response following therapy.Analysis on tissue array samples for which clinical and Herceptest datawas available and who over-expressed HER2/neu.

TABLE 5 patient group n % responders % relapse P value EGFR pos/p-ERKpos 21 14% 86% 0.04 EGFR pos/p-ERK neg 19 42% 58% EGFR neg/p-ERK pos 90% 100% EGFR neg/p-ERK neg 14 14% 86% EGFR pos/p-AKT pos 17 18% 82% 0.07EGFR pos/p-AKT neg 26 38% 62% EGFR neg/p-AKT pos 5 20% 80% EGFRneg/p-AKT neg 18 6% 94% IGFR pos/p-S6 pos 13 8% 92% 0.01 IGFR pos/p-S6neg 20 35% 65% IGFR neg/p-S6 pos 12 67% 33% IGFR neg/p-S6 neg 23 26% 74%Analysis of receptor and downstream protein activation versus responsein patients following therapy. Analysis on tissue array samples forwhich clinical and Herceptest data was available and who over-expressedHER2/neu.

TABLE 6 % patient group n responders % relapse P value NDF neg/p-S6pos/IGFR neg 2 50% 50% 0.003 NDF neg/p-S6 neg/IGFR neg 9 11% 89% NDFneg/p-S6 neg/IGFR pos 4 0% 100% NDF neg/p-S6 pos/IGFR pos 4 0% 100% NDFpos/p-S6 pos/IGFR neg 7 100% 0% NDF pos/p-S6 neg/IGFR pos 16 44% 56% NDFpos/p-S6 neg/IGFR neg 14 36% 64% NDF neg/p-ERK pos/EGFR neg 3 0% 100%0.08 NDF neg/p-ERK neg/EGFR neg 4 0% 100% NDF neg/p-ERK neg/EGFR pos 1020% 80% NDF neg/p-ERK pos/EGFR pos 6 0% 100% NDF pos/p-ERK pos/EGFR neg5 0% 100% NDF pos/p-ERK neg/EGFR pos 13 54% 46% NDF pos/p-ERK neg/EGFRneg 6 17% 83% NDF pos/p-ERK pos/EGFR pos 18 28% 72%Analysis of ligand and receptor expression and downstream proteinactivation versus patient response in patients following therapy.Analysis on tissue array samples for which clinical and Herceptest datawas available and who over-expressed HER2/neu.

TABLE 7 Patient IGFR EGFR p-S6 p-AKT p-ERK Response #1 + + − − − yes #2− + + + + yes #3 + + − + − yes #4 + − + + + no #5 + + + + − no #6 +− + + − no #7 + + + + + noReceptor tyrosine kinase expression, downstream protein activation andpatient response to therapy in seven breast cancer patients. Analysiswas of whole tissue sections.

TABLE 8  AKT (NP_005154 GI:4885061) 480 AMINO ACIDS (SEQ ID NO: 1)See, e.g., Staal, S.P., Proc. Natl. Acad. Sci. U.S.A. 84(14), 5034-5037 (1987).MSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKKQEEEEMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASSTA (SEQ ID NO: 1)S6 (NP_001001, GI:17158044) 249 AMINO ACIDS (SEQ ID NO: 2)See, e.g., Pata et al., (1992) Gene 121 (2), 387-392.MKLNISFPATGCQKLIEVDDERKLRTFYEKRMATEVAADALGEEWKGYVVRISGGNDKQGFPMKQGVLTHGRVRLLLSKGHSCYRPRRTGERKRKSVRGCIVDANLSVLNLVIVKKGEKDIPGLTDTTVPRRLGPKRASRIRKLFNLSKEDDVRQYVVRKPLNKEGKKPRTKAPKIQRLVTPRVLQHKRRRIALKKQRTKKNKEEAAEYAKLLAKRMKEAKEKRQEQIAKRRRLSSLRASTSKSESSQK (SEQ ID NO: 2)ERK (XP_055766, GI:20562757) 379 AMINO ACIDS (SEQ ID NO: 3)See, e.g., Butch et al., J Biol Chem., 1996., 271 (8): 4230-5.MAAAAAQGGGGGEPRRTEGVGPGVPGEVEMVKGQPFDVGPRYTQLQYIGEGAYGMVSSAYDHVRKTRVAIKKISPFEHQTYCQRTLREIQILLRFRHENVIGIRDILRASTLEAMRDVYIVQDLMETDLYKLLKSQQLSNDHICYFLYQILRGLKYIHSANVLHRDLKPSNLLINTTCDLKICDFGLARIADPEHDHTGFLTEYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKHYLDQLNHILGILGSPSQEDLNCIINMKARNYLQSLPSKTKVAWAKLFPKSDSKALDLLDRMLTFNPNKRITVEEALAHPYLEQYYDPTDEPVAEEPFTFAMELDDLPKERLKELIFQETARFQPGVLEAP (SEQ ID NO: 3)

We claim:
 1. A method for identifying a breast tumor likely to respondto HER-2 directed therapy, comprising: measuring by immunohistochemistryin a tissue sample obtained from the breast tumor: a) expression ofinsulin-like growth factor receptor (IGFR) polypeptide; and b)phosphorylation of S6 ribosomal polypeptide; and identifying the breasttumor as a breast tumor likely to respond to HER-2 directed therapy, ifthe breast tumor is negative for IGFR polypeptide expression andpositive for phosphorylation of S6 ribosomal polypeptide.
 2. The methodof claim 1, wherein the HER2-directed therapy comprises trastuzumab. 3.The method of claim 1, wherein the tissue sample is a biopsy sample. 4.The method of claim 3, wherein the biopsy sample is a paraffin-embeddedsample.
 5. The method of claim 1, wherein phosphorylation of S6ribosomal polypeptide is determined using an antibody specific for anepitope comprising a phosphorylated serine at position 235 of SEQ ID NO:2.
 6. The method of claim 1, wherein the breast tumor overexpresses HER2polypeptide.
 7. The method of claim 1, wherein the breast tumor haspreviously been treated with at least one chemotherapeutic.
 8. Themethod of claim 7, wherein the at least one chemotherapeutic comprisescisplatin, doxorubicin, or taxol.
 9. The method of claim 1, furthercomprising administering the HER-2 directed therapy to the subject fromwhich the breast tumor was obtained if the breast tumor is negative forIGFR polypeptide expression and positive for phosphorylation of S6ribosomal polypeptide.
 10. A method for identifying a breast tumorlikely to respond to HER-2 directed therapy, comprising: measuring in atissue sample obtained from the breast tumor: a) expression ofinsulin-like growth factor receptor (IGFR) polypeptide; and b)phosphorylation of S6 ribosomal polypeptide; identifying the breasttumor as a breast tumor likely to respond to HER-2 directed therapy, ifthe breast tumor is negative for IGFR polypeptide expression andpositive for phosphorylation of S6 ribosomal polypeptide; andadministering a HER-2 directed therapy to the subject from which thebreast tumor sample was obtained if the breast tumor is negative forIGFR polypeptide expression and positive for phosphorylation of S6ribosomal polypeptide.
 11. The method of claim 10, wherein theHER2-directed therapy comprises trastuzumab.
 12. The method of claim 10,wherein the tissue sample is a biopsy sample.
 13. The method of claim12, wherein the biopsy sample is a paraffin-embedded sample.
 14. Themethod of claim 10, wherein phosphorylation of S6 ribosomal polypeptideis determined using an antibody specific for an epitope comprising aphosphorylated serine at position 235 of SEQ ID NO:
 2. 15. The method ofclaim 10, wherein the breast tumor overexpresses HER2 polypeptide. 16.The method of claim 10, wherein the breast tumor has previously beentreated with at least one chemotherapeutic.
 17. The method of claim 16,wherein the chemotherapeutic comprises cisplatin, doxorubicin, or taxol.